A. We hypothesized that factors involved in controlling PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, TNAP, would be involved in root formation, cementogenesis. a. Teeth obtained from Alpl KO mice, beyond cementum phenotype (no acellular cementum, altered PDL, exfoliation of teeth) exhibited dentin/enamel phenotype, mimicking humans. Enamel, dentin and cementum defects were corrected by TNAP enzyme replacement, mice and humans (latter by Millan's group independently) b. Healing, using a periodontal defect wound model, occurred more rapidly in Ank KO mice, demonstrating significance of decreasing PPi levels at local sites toward promoting periodontal tissue repair c. Teeth from AlplxPhospho1 dKO mice exhibited a more profound dentin phenotype than comparable tissues from KO of Alpl or Phospho1 demonstrating the function of PHOSPHO1 in controlling Pi levels in matrix vesicles (MVs), and TNAP and PHOSPHO1 act synergistically in regulating mineralization. Recent data, in collaboration with Millans group, suggest that PHOSPHO1 plays a role in cementum formation, an unexpected finding. Thus, either PHOSPHO1 has a role in regulating Pi/PPi beyond MVs and/or cementum formation involves MVs d. In vitro, ANK and NPP1 were shown to be mineralized-driven genes/protein and coupled with in vivo findings, demonstrated the importance of Pi/PPi modulation for cementogenesis and provide the rationale for ongoing studies using in vitro methods to identify small molecules controlling mineralization and in vivo periodontal wound healing models to deliver factors controlling Pi/PPi, locally and systemically e. Intriguingly, examining periodontal tissues from Ank and Npp1 KO animals demonstrated a compensatory mechanism related to gene expression, ENPP1 protein levels increased in cementum in Ank KO tissues, while ANK protein levels increased in Npp1 KO tissues, yet the cementum phenotype was not altered, most likely due to the profound role of TNAP in providing Pi at local sites f. Cementum from of Npp1 or Ank KO mice exhibited a marked increase in protein expression for both OPN and DMP1, and further cementoblasts exposed to Pi in vitro also exhibited an increase in transcripts for these two genes. These data provide a unified hypothesis for how Pi/PPi ratio developmentally tunes cementum formation and properties, an insight that we plan to employ to stimulate cementum regeneration, using murine models. B. We hypothesized a relationship exists between the SIBLING family, e.g. BSP, OPN and DMP1, their associated integrins, and key regulators of phosphate metabolism and modulating SIBLINGS provides therapeutic strategy to regenerate cementum. a. Bone sialoprotein affects cell attachment and signaling through an RGD integrin-binding region, and acts as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. To test our hypothesis we analyzed tooth development in a Bsp null (-/-) mouse model. Developmental analysis, in collaboration with Goldbergs group, by histology, histochemistry, and SEM revealed a marked reduction in acellular cementum formation on Bsp -/- teeth, and cementum deposited was hypomineralized.. Loss of BSP caused disorganized PDL and increased epithelial down-growth. Bsp -/- mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and osteoclasts. Increased staining for DMP1 in the region of acellular formation in tissues from KO mice vs. WT tissues was noted. Results suggest that BSP plays an essential and non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. We are exploring the integrin/BSP relationship with Fisher at NIDCR b. In a related project with Young, NIDCR, we hypothesized that alterations in proteoglycans would compromise periodontal tissues and expression of key SIBLINGS. dKO for two SLRPs, fibromodulin and biglycan, acquire ectopic calcification of tendons and severe osteoarthritis. Polarized light studies demonstrated abnormal collagen fibrils and defective remodeling of alveolar bone of dKO mice. IHC revealed increased staining of SLRPs, asporin, lumican and decorin, in PDL of dKO mice, indicating compensatory mechanisms. DMP1, considered to be mechanosensory osteocyte marker, was increased markedly in dKO PDL. Disruption of homeostasis of dKO PDL was further supported by increased expression of RANKL and elevated numbers of TRAP positive osteoclasts. To elucidate underlying mechanisms, PDL tissues from dKO mice were analyzed by PCR array. Results indicated hyperactive TGF beta/BMP signaling in dKO tissues with significant increased expression of SMAD-4 and -5 genes, and elevated staining of phosphorylated SMAD5 in dKO PDL. Therefore we propose that Fmod and Bgn sequester BMP preventing binding to BMP receptors. The predicted outcome of deletion of Fmod and Bgn from ECM would be an elevated sensitivity to TGF beta/BMP which could affect BMPs signal transduction, causing an increased expression of phosphorylated SMAD5 in PDL. C. We hypothesized that the ability of PTH and 1,25D to modulate FGF23 expression involves an intermediate factor and specifically, DMP1. 1,25D: Murine cementoblasts and osteocyte-like cells were used. Dmp1 mRNA levels decreased by 50% in the presence of 1,25D, while use of a VDR agonist and antagonist confirmed that vitamin D receptor pathway activation was required. Further analysis showed that histone deacetylase (HDAC) recruitment was necessary. PTH: Cementoblasts and cementocytes exposed to PTH (1-34) exhibited a decrease in expression of Dmp1 mRNA in a dose-response and time-dependent manner. Western blot detected a 30% down-regulation of DMP1 with PTH treatment.Treatment with PTH increased intracellular cAMP levels by 10-fold compared to control, and forskolin, a cAMP/PKA pathway agonist, also decreased Dmp1 mRNA expression, implicating the cAMP/PKA pathway. Our results suggest that PTH may modulate FGF23 via several pathways e.g. SOST as well as DMP-1 and further that PTH and 1,25D-mediated effects on FGF23 may share some common mechanistic aspects. Future studies using RNAseq approaches will help determine the specific DNA sequences and transcription factor(s) involved in Dmp1 regulation. Research related to the DMP1 promoter has been in collaboration with Collins at NIDCR and Presland at UW.